1. Field of the Invention
The present invention relates to Chlamydia psittaci vaccines and to methods of protecting animals, including avian species, from Chlamyida psittaci infections.
2. Background Art
The genus Chlamydia contains four species of obligate parasitic bacteria: Chlamydia psittaci, Chlamydia pecorum, Chlamydia pneumoniae, and Chlamydia trachomatis. This unique genus causes a variety of diseases in humans, mammals, and birds. In humans, the most notable are trachoma and urogenital infections due to C. trachomatis and psittacosis caused by C. psittaci. In animals, C. psittaci can cause a diverse range of disease in livestock, poultry, turkeys and companion birds. The known C. psittaci strains have been grouped into eight biovars (Perez-Martinez, J A and J Storz, 1985). Strains of serovar 1 are mainly associated with intestinal infections and abortions, while strains of serovar 2 cause polyarthritis, encephalitis, and conjunctivitis in ruminants. Avian strains of C. psittaci cause respiratory problems and diarrhea in birds (Storz, 1988). The organism can also be transmitted to humans from these animals, and outbreaks have been documented in animal production workers. Thus, there is a need for an effective vaccine against C. psittaci for mammalian and avian species.
The chlamydia organism goes through two developmental stages in its life cycle. The extracellular form, which is the infectious entity of the cycle, is called the elementary body (EB). These EBs attach and enter the host cell, where they re-organize into reticulate bodies (RBs) which divide within membrane-bound host cell compartments by binary fission and then condense into a new generation of infectious EBs. The attachment and entry of the EB into the host cell is a receptor-mediated phenomenon (Hodinka et al. 1988), and several chlamydial proteins have been implicated in the EB attachment to host cellular membranes (Baghian and Schnorr, 1992). One of these proteins is called the xe2x80x9cmajor outer membrane proteinxe2x80x9d, or MOMP, and surface-exposed epitopes of this protein from C. trachomatis have been shown to block EB attachment onto the host cell (Su and Caldwell, 1991). The MOMP genes from some strains of C. psittaci and C. trachomatis have been sequenced (Baehr et al., 1988, Pickett et al. 1988, Yuan et al. 1989, Zhang et al. 1989, Kaltenboeck, et al. 1993). Analyses of these sequences revealed that portions of the structure of this protein are conserved between species. There are also four regions of xe2x80x9cvariable domainxe2x80x9d interspersed with conserved sequences, and these are referred to as VD1, VD2, VD3, and VD4. The location of these VD regions are identical in the two species (see Zhang et al., 1989). A comparison of the genes encoding the MOMP from C. psittaci and C. trachomatis show that, overall, the sequences are approximately 68% identical.
In C. trachomatis, these four variable regions have been shown to be involved in the neutralization of EB infectivity, in serotype specificity, (Baehr, et al. 1988; Peeling et al. 1984; and Spears and Storz 1979) as well as in the pathogenicity of the strains (Baehr et al. 1988 and Su et al. 1988). Nonetheless, the development of subunit vaccines for C. trachomatis has been hampered by the difficulty in expressing the native, full-length MOMP gene in a recombinant vector host (Manning and Stewart, 1993). There is no known published work on the expression of the C. psittaci MOMP gene prior to that described herein. Consequently, there remains a need to develop an effective subunit vaccine for animal and avian species to protect them from C. psittaci infections.
The present invention provides a vaccine composition which is protective against Chlamydia psittaci infections in animals, including avian species, comprising an immunogenic amount of a C. psittaci major outer membrane protein (MOMP) polypeptide lacking regions VD1 and VD2. Also provided are polypeptides and isolated nucleic acids encoding such polypeptides, as well as methods of preventing C. psittaci infections in animals comprising administering to the subject animal such vaccine compositions.
Also provided are nucleic acid vectors for the expression of a MOMP polypeptide-MBP fusion protein comprising a nucleic acid encoding MBP and a nucleic acid encoding an immunogenic C. psittaci MOMP polypeptide arranged in tandem such that the MOMP-MBP fusion protein can be expressed.
Additionally provided are methods of preventing a Chlamydia psittaci infection in a subject comprising administering to the subject a nucleic acid vaccine comprising an immunizing amount of a nucleic acid vector comprising a nucleic acid encoding an immunogenic C. psittaci MOMP polypeptide lacking regions VD1 and VD2.
Definitions
xe2x80x9cMOMPxe2x80x9d means the major outer membrane protein from a Chlamydia psittaci strain.
As used herein, the term xe2x80x9cpolypeptidexe2x80x9d refers to a polymer of amino acids. As used in combination with MOMP, it means a fragment of MOMP.
As used in the specification and in the claims, xe2x80x9caxe2x80x9d can mean one or more, depending upon the context in which it is used.
Maltose Binding Protein, or xe2x80x9cMBPxe2x80x9d means a maltose binding protein.
The term xe2x80x9cimmunogenic amountxe2x80x9d means an amount of an immunogen, i.e., a MOMP polypeptide, a MOMP polypeptide-MBP fusion protein, or portions thereof, which is sufficient to induce an immune response in a vaccinated animal and which protects the animal against active infection with Chlamydia psittaci upon exposure thereto.
The term xe2x80x9cimmunizing amountxe2x80x9d means an amount of a nucleic acid expression vector sufficient to induce an immune response in a vaccinated animal and which protects the animal against active infection with Chlamydia psittaci upon exposure thereto.
Detailed Description
Vaccine preparations that are efficacious and economical for use in human and non-human animals, including birds, such as chickens, turkeys and companion birds, are provided.
Polypeptide Vaccines
The present invention provides a vaccine composition which is protective against Chlamydia psittaci infections in animals, including avian species, comprising an immunogenic amount of a C. psittaci major outer membrane protein (MOMP) polypeptide lacking variable regions VD1 and VD2. In specific embodiments, the vaccine composition can comprise an immunogenic amount of a C. psittaci major outer membrane protein (MOMP) polypeptide comprising amino acids 183 through 402 of the MOMP protein from C. psittaci strains Avian Type C, LSUWTCK (a strain isolated from a cockatiel which has the identical MOMP gene sequence to Avian Type C), or strain 6BC (which is identical to the sequence of Mn except for a single amino acid change), or amino acids 164 to 389 of the MOMP protein from C. psittaci strain B577.
The complete nucleic acid sequences of the MOMP gene from C. psittaci strains Avian Type C, B577, and 6BC are provided herein as SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 13, respectively, and the corresponding amino acid sequences of the MOMP proteins are provided herein as SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14, respectively.
The MOMP polypeptide is purified from other proteins sufficiently to induce a specific immune response. Thus, in specific embodiments, the MOMP polypeptide comprising the vaccine comprises the amino acid sequence set forth in SEQ ID NO:1. In another embodiment, the MOMP polypeptide of the vaccine comprises the amino acid sequence set forth in SEQ ID NO:2.
In a specific embodiment of this invention, the MOMP polypeptide is provided as a component of a fusion protein. Thus the present invention provides a vaccine composition comprising a MOMP polypeptide, as described herein, linked to a non-MOMP polypeptide or protein, and a nucleic acid encoding such a fusion protein. A MOMP fusion protein can be made with any desired protein as part of the chimera. One example is glutathione-S-transferase (xe2x80x9cGSTxe2x80x9d) which is commonly used as a fusion protein. Another example is Maltose Binding Protein (MBP). Thus, in one embodiment, a vaccine composition comprises a Maltose Binding Protein-MOMP fusion protein, wherein the MOMP portion of the fusion protein is a C. psittaci MOMP polypeptide. Such a fusion protein can have MBP as the amino terminal protein of the fusion protein and the MOMP polypeptide as the carboxyl terminal portion of the fusion protein. Specifically provided is a vaccine composition comprising an MBP-MOMP fusion protein which includes the polyamino acid encoded by nucleotides 1606-2661 of the MBP sequence from the E. coli malE gene (available, for example, in the vector pMAL(trademark)-c2 from New England Biolabs, Inc., Beverly, Mass. 01915-5999). Also specifically provided is a vaccine composition comprising MBP-MOMP fusion protein wherein the MOMP polypeptide is a C. psittaci MOMP polypeptide comprising amino acids 183 through 402 of the MOMP protein from either C. psittaci strains Avian Type C, LSUWTCK, or 6BC, or amino acids 164 to 389 of the MOMP protein from C. psittaci strain B577. Fusion proteins utilizing MBP sequences are presented in U.S. Pat. No. 5,643,758. The fusion protein of this invention has several advantageous characteristics. The unexpected discovery that such a MBP-MOMP fusion protein precipitates in inclusion bodies in the bacterial host cells provided an economical method for purifying this immunogen. Specifically, for example, this MBP-MOMP fusion protein can be expressed from a nucleic acid encoding it in relatively large amounts (e.g., 100 milligrams per liter of E.coli) in a manner such that the fusion protein can be very easily purified to a useful extent. Typically, MBP-protein fusions are purified by passing the cell extract over an affinity column bound with an appropriate ligand for MBP. The added cost of such a preparation step generally makes such proteins uneconomical as vaccines in production animals. The MBP-MOMP fusion protein of this invention can be prepared to sufficient purity without the use of such a column, making the economical production of a production or companion animal vaccine possible. This MBP-MOMP fusion protein precipitates as inclusion bodies, and thus traditional purification techniques, such as affinity columns, are not necessary, and the resulting purified fusion protein retains immunogenicity.
Polypeptides having amino acid substitutions from the sequences set forth, that do not significantly reduce the immunogenicity of the polypeptides, are contemplated by this invention. For example, amino acid substitutions can be selected by known parameters to be neutral substitutions (see, e.g., Robinson W E Jr, and Mitchell W M., AIDS 4:S151-S162(1990)). As will be appreciated by those skilled in the art, the invention also includes those polypeptides having slight variations in amino acid sequences or other properties. Such variations may arise naturally as allelic variations (e.g., due to genetic polymorphism) or may be produced by human intervention (e.g., by mutagenesis of cloned DNA sequences), such as induced point, deletion, insertion and substitution mutants. Minor changes in amino acid sequence are generally preferred, such as conservative amino acid replacements, small internal deletions or insertions, and additions or deletions at the ends of the molecules. Substitutions may be designed based on, for example, the model of Dayhoff, et al. (in Atlas of Protein Sequence and Structure 1978, Nat""l Biomed. Res. Found., Washington, D.C.). These modifications can result in changes in the amino acid sequence, provide silent mutations, modify a restriction site, or provide other specific mutations. Additionally, the amino acid sequences of the MOMP polypeptide vaccines of this invention can include sequences in which one or more amino acids have been substituted with another amino acid to provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to increase its bio-longevity, alter enzymatic activity, or alter interactions with gastric acidity. In any case, the peptide must retain C. psittacixe2x80x94protective immunogenicity.
It is also contemplated in this invention that the variable regions VD3 and VD4 can be administered either in the form of a single MOMP polypeptide, for example SEQ ID NO:1 or SEQ ID NO:2, or as multiple MOMP polypeptides, each one encoding one of the variable regions. Techniques for producing such MOMP polypeptides are routine in art, given the knowledge of the full DNA sequence of the MOMP genes. For example, one can construct multiple nucleic acid vectors by first enzymatically cleaving the DNA of the MOMP gene at restriction enzyme sites located on either side and between variable regions VD3 and VD4 and then cloning the resulting DNA fragments into appropriate vectors for expression. Alternatively, as shown in the Examples herein, artificial primers can be designed to amplify the desired portions of the MOMP gene with convenient restriction enzyme recognition sites in the primers, to allow rapid and efficient cloning of selected portions of the MOMP gene into expression vectors.
Selected MOMP polypeptides can be assayed for immunogenicity and specificity. Briefly, various concentrations of a putative immunogenically specific polypeptide are prepared and administered to an animal and the immunological response (e.g., the production of antibodies or cell mediated immunity) of an animal to each concentration is determined. The amounts of antigen administered depend on the subject, e.g. a human or a non-human animal, including a bird, the condition of the subject, the size of the subject, etc. Thereafter an animal so inoculated with the antigen can be exposed to the bacterium to test the potential vaccine effect of the specific immunogenic MOMP polypeptide. The specificity of a putative immunogenic MOMP polypeptide can be ascertained by testing sera, other fluids or lymphocytes from the inoculated animal for cross-reactivity with other closely related bacteria. Nucleic acids The present invention provides an isolated nucleic acid comprising a nucleic acid which encodes a C. psittaci MOMP polypeptide lacking VD1 and VD2. In a specific embodiment, the nucleic acid includes the variable regions VD3 and VD4 (e.g., amino acids 183 to 402 of the C. psittaci Avian Type C, LSUWTCK, or 6BC MOMP protein, or amino acids 162 to 389 of the C. psittaci B577 MOMP protein. Additionally, the amino terminus end of the isolated nucleic acid can be modified from the native sequence to include an ATG (methionine) start codon and a Kozak regulatory sequence, both of which are typically required for translation in eukaryotes.
In a specific embodiment, the present invention provides an isolated nucleic acid comprising a nucleic acid having the nucleotide sequence set forth in SEQ ID NO:3. This nucleic acid encodes roughly the carboxyl-terminal half of the C. psittaci strain Avian Type C MOMP protein and includes VD3 and VD4. Additionally, the amino terminus end of this sequence has been modified from the native sequence to include an ATG (methionine) start codon and a Kozak regulatory sequence. Another embodiment of this invention provides an isolated nucleic acid having the nucleotide sequence set forth in SEQ ID NO: 4, which ends at the stop codon TAA, and thus does not include the 3xe2x80x2 untranslated sequences included in SEQ ID NO:3.
In another embodiment, the present invention provides an isolated nucleic acid comprising a nucleic acid having the nucleotide sequence set forth in SEQ ID NO: 5. This nucleic acid encodes roughly the carboxyl-terminal half of the C. psittaci strain B577 MOMP protein and includes VD3 and VD4, and additionally has an ATG (methionine) and a Kozak consensus sequence at the amino terminus of this polypeptide. Another embodiment of this invention provides an isolated nucleic acid having the nucleotide sequence set forth in SEQ ID NO: 6, which ends at the stop codon TAA, and thus does not include the 3xe2x80x2 untranslated sequences included in SEQ ID NO:5.
The present invention also provides a composition which is protective against C. psittaci infections comprising polypeptides expressed in a suitable host by one or more of the isolated nucleic acids of this invention and a pharmaceutically acceptable carrier.
Also provided is a nucleic acid vector for the expression of a MOMP polypeptide-MBP fusion protein comprising a nucleic acid encoding MBP and a nucleic acid encoding a MOMP polypeptide arranged in tandem such that the MOMP-MBP fusion protein can be expressed. In a specific embodiment, the nucleic acid encodes an amino acid sequence for the MOMP polypeptide selected from the group consisting of: SEQ ID NO:1 and SEQ ID NO:2. In a preferred embodiment, the nucleic acid encoding MBP is located 5xe2x80x2 to the nucleic acid encoding MOMP. Persons skilled in the art are knowledgeable in arranging the nucleic acids such that the fusion protein can be expressed, including ensuring that the reading frame for both nucleic acids is the same, and including various control regions, as also further discussed herein.
In another embodiment, the present invention provides a nucleic acid vector for the transient expression of a C. psittaci MOMP polypeptide in a eukaryotic cell comprising a eukaryotic promoter functionally linked to a nucleic acid encoding a C. psittaci MOMP polypeptide lacking regions VD1 and VD2. In a specific embodiment, the nucleic acid encodes an amino acid sequence selected from the group consisting of: SEQ ID NO:7 or SEQ ID NO:8. In a further specific embodiment, the nucleic acid has a sequence selected from the group consisting of: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. Any desired eukaryotic promoter can be utilized; however, preferable promoters are those that are strong promoters in avian or mammalian cells, as are known in the art and further described below. As discussed herein, certain modifications to the nucleic acid vectors can be made.
In a specific embodiment, provided herein is a nucleic acid vector for the transient expression of a C. psittaci MOMP polypeptide in a eukaryotic cell comprising a cytomegalovirus promoter functionally linked to a nucleic acid encoding a C. psittaci MOMP polypeptide lacking regions VD1 and VD2. In a further specific embodiment, the nucleic acid encodes an amino acid sequence selected from the group consisting of: SEQ ID NO:7 and SEQ ID NO:8.
Nucleic Acid Vaccines
The present invention provides compositions comprising a plurality of the nucleic acid vectors for the transient expression of a C. psittaci MOMP polypeptide in a eukaryotic cell comprising a eukaryotic promoter functionally linked to a nucleic acid encoding amino acid sequence comprising a C. psittaci major outer membrane protein (MOMP) polypeptide lacking regions VD1 and VD2. Such compositions ate administered to a subject such that they can be expressed in the subject, the xe2x80x9cpluralityxe2x80x9d of vectors being sufficient to induce an immune response.
In a specific embodiment, the present invention provides a composition which is protective against a Chlamydia psittaci infection in animals, including avian species, comprising a plurality of nucleic acid expression vectors comprising a eukaryotic promoter functionally linked to a nucleic acid encoding the amino acid sequence set forth in SEQ ID NO:7 or SEQ ID NO:8 in a pharmaceutically acceptable carrier.
In a further specific embodiment, the present invention provides a composition which is protective against Chlamydia psittaci infections in animals, including avian species, comprising a plurality of nucleic acid expression vectors comprising a eukaryotic promoter functionally linked to one or more nucleotide sequences selected from the group consisting of: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 in a pharmaceutically acceptable carrier.
In a specific embodiment, the nucleic acid expression vector comprises a cytomegalovirus promoter functionally linked to a nucleic acid encoding VD3 and VD4 of MOMP, for example having the nucleotide sequence set forth in either SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.
Nucleic Acids/Vectors/Vaccines-general
The nucleic acid encoding the MOMP polypeptide can be any nucleic acid that functionally encodes the MOMP polypeptide. For example, to functionally encode, i.e., allow the nucleic acid to be expressed, the nucleic acid can include, for example, expression control sequences, such as an origin of replication, a promoter, an enhancer, and necessary information processing sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Preferred expression control sequences are strong and/or inducible promoters such as those derived from metallothionine genes, actin genes, immunoglobulin genes, CMV, SV40, Rous sarcoma virus, adenovirus, bovine papilloma virus, etc. A nucleic acid encoding a selected MOMP polypeptide can readily be determined based upon the genetic code for the amino acid sequence of the selected MOMP polypeptide, and, clearly, many nucleic acids will encode any selected chimeric protein. Modifications to the nucleic acids of the invention are also contemplated, since mutations can thereby be studied for greater protective vaccine effect. Additionally, modifications that can be useful are modifications to the sequences controlling expression of the MOMP polypeptide to make production of the MOMP polypeptide inducible or repressible upon addition to the cells of the appropriate inducer or repressor. Such means are standard in the art (see, e.g.,. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989). The nucleic acids can be generated by means standard in the art, such as by recombinant nucleic acid techniques, as exemplified in the examples herein, and by synthetic nucleic acid synthesis or in vitro enzymatic synthesis.
The expression vectors of the invention can be in a host capable of expressing the MOMP polypeptide immunogen or the MBP-MOMP fusion protein immunogen. There are numerous E. coli expression vectors known to one of ordinary skill in the art useful for the expression of the antigen. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilus, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts one can also make expression vectors, which will typically contain expression control sequences compatible with the host cell (e.g., an origin of replication). In addition, any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (Trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda. The promoters will typically control expression, optionally with an operator sequence, and have ribosome binding site sequences for example, for initiating and completing transcription and translation. If necessary an amino terminal methionine can be provided by insertion of a Met codon 5xe2x80x2 and in-frame with the antigen. Also, the carboxyl-terminal extension of the antigen can be removed using standard oligonucleotide mutagenesis procedures.
Additionally, yeast expression systems can be used. There may be several advantages to yeast expression systems. First, evidence exists that proteins produced in a yeast secretion systems exhibit correct disulfide pairing. Second, post-translational glycosylation is efficiently carried out by yeast secretory systems. The Saccharomyces cerevisiae pre-pro-alpha-factor leader region (encoded by the MF xcex1-1 gene) is routinely used to direct protein secretion from yeast (Brake et al., 1984). The leader region of pre-pro-alpha-factor contains a signal peptide and a pro-segment which includes a recognition sequence for a yeast protease encoded by the KEX2 gene: this enzyme cleaves the precursor protein on the carboxyl side of a Lys-Arg dipeptide cleavage-signal sequence. The antigen coding sequence can be fused in-frame to the pre-pro-alpha-factor leader region. This construct is then put under the control of a strong transcription promoter, such as the alcohol dehydrogenase I promoter or a glycolytic promoter. The antigen coding sequence is followed by a translation termination codon which is followed by transcription termination signals. Alternatively, the antigen coding sequences can be fused to a second protein coding sequence, such as Sj26 or xcex2-galactosidase, used to facilitate purification of the fusion protein by affinity chromatography. The insertion of protease cleavage sites to separate the components of the fusion protein is applicable to constructs used for expression in yeast.
Additionally, mammalian cells permit the expression of proteins in an environment that favors important post-translational modifications such as folding and cysteine pairing, addition of complex carbohydrate structures, and secretion of active protein. Vectors useful for the expression of antigen in mammalian cells are characterized by insertion of the antigen coding sequence between a strong viral promoter and a polyadenylation signal. The vectors can contain genes conferring either gentamicin or methotrexate resistance for use as selectable markers. The antigen and immunoreactive fragment coding sequence can be introduced into a Chinese hamster ovary cell line using a methotrexate resistance-encoding vector. Presence of the vector DNA in transformed cells can be confirmed by Southern analysis and production of an RNA corresponding to the antigen coding sequence can be confirmed by Northern analysis. A number of other suitable host cell lines capable of secreting intact human proteins have been developed in the art, and include the CHO cell lines, HeLa cells, myeloma cell lines, Jurkat cells, etc. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer, and necessary information processing sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, Adenovirus, Bovine Papilloma Virus, etc. The vectors containing the DNA segments of interest can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cellular hosts.
Alternative vectors for the expression of antigen in mammalian cells, those similar to those developed for the expression of human gamma-interferon, tissue plasminogen activator, clotting Factor VIII, hepatitis B virus surface antigen, protease Nexinl, and eosinophil major basic protein, can be employed. Further, the vector can include cytomegalovirus (CMV) promoter sequences and a polyadenylation signal available for expression of inserted DNAs in mammalian cells.
The nucleic acid vectors for transient expression in a eukaryotic cell are suitable for genetic, or xe2x80x9cnaked nucleic acidxe2x80x9d, immunization. These can be constructed using any of a variety of eukaryotic promoters, herein also referred to as cis-acting transcription/translation regulatory sequences, known in the art. General methods for the construction, production and administration of nucleic acid vaccines are known in the art, e.g. Vogel, FR and N Sarver (1995) Clin. Microbiol. Rev. 8:406-410.
These nucleic acid vectors comprise nucleic acids that functionally encode, i.e. are functionally linked to a nucleic acid encoding a MOMP polypeptide. For example, to functionally encode, i.e., allow the nucleic acid to be expressed, the nucleic acid can include, for example, expression control sequences, such as a cis-acting transcription/translation regulatory sequence (comprising one or more of the following: a promoter, response element(s), an initiator sequence), an enhancer, and information processing sites, such as ribosome binding sites, RNA splice sites, intron elements, polyadenylation sites, and transcriptional terminator sequences, all of which, either alone or in combinations, are capable of directing expression in the target animal. Preferred expression control sequences are strong and/or inducible cis-acting transcription/translation regulatory sequences such as those derived from metallothionine genes, actin genes, myosin genes, immunoglobulin genes, cytomegalovirus (CMV), SV40, Rous sarcoma virus, adenovirus, bovine papilloma virus, etc. The C. psittaci MOMP-encoding nucleic acid and expression control sequences are constructed in a vector, such as a plasmid of bacterial origin, for administration to the target animal. There are numerous plasmids known to those of ordinary skill in the art useful for the production of nucleic acid vaccine plasmids. A specific embodiment employs constructs using the plasmid xe2x80x9cpcDNA3.1+xe2x80x9d as the vector (InVitrogen Corporation, Carlsbad, Calif.). In addition, the nucleic acid expression vectors that functionally encode a MOMP polypeptide may additionally contain immunostimulatory sequences (xe2x80x9cISSxe2x80x9d) that stimulate the animals"" immune system. Other possible additions to the nucleic acid expression vectors include nucleic acid sequences encoding cytokines, such as granulocyte macrophage colony stimulating factor (GM-CSF) or interleukin-12 (IL-12). The cytokines can be used in various combinations to fine-tune the response of the animal""s immune system, including both antibody and cytotoxic T lymphocyte responses, to bring out the specific level of response needed to protect the animal from the targeted disease.
Alternatively, the nucleic acid expression vectors can be constructed in a non-replicating retroviral vector, such as the Moloney murine leukemia virus (N2) backbone described by Irwin, et al. (1994, J. Virology 68:5036-5044).
The present genes were isolated from C. psittaci; however, homologs from any Chlamydia strain infecting a selected species, can readily be obtained by screening a library from that Chlamydia strain, genomic or cDNA, with a probe comprising sequences of the nucleic acids set forth in the sequence listing herein, or fragments thereof, and isolating genes specifically hybridizing with the probe under preferably relatively high stringency hybridization conditions. For example, high salt conditions and/or high temperatures of hybridization can be used. For example, the stringency of hybridization is typically about 5xc2x0 C. to 20xc2x0 C. below the Tm (the melting temperature at which half of the molecules dissociate from its partner) for the given chain length. As is known in the art, the nucleotide composition of the hybridizing region factors in determining the melting temperature of the hybrid. For 20mer probes, for example, the recommended hybridization temperature is typically about 55-58xc2x0 C. Additionally, the C. psittaci MOMP sequence can be utilized to devise a probe for a homolog in any specific animal by determining the amino acid sequence for a portion of the C. psittaci protein, and selecting a probe with optimized codon usage to encode the amino acid sequence of the homolog in that particular animal.
The present invention contemplates cells containing a nucleic acid expression vector of the invention. A cell containing a nucleic acid expression vector encoding a MOMP polypeptide typically can replicate the DNA and, further, typically can express the encoded MOMP polypeptide. The cell can be a prokaryotic cell, particularly for the purpose of producing quantities of the nucleic acid, or a eukaryotic cell, particularly a mammalian cell. The cell is preferably a mammalian cell for the purpose of expressing the encoded protein so that the resultant produced protein has mammalian protein processing modifications. The cell also can be a eukaryotic cell in a host organism, for the purposes of vaccinating the host via genetic or xe2x80x9cnaked nucleic acidxe2x80x9d immunization. In the case of genetic immunization, the nucleic acid expression vector does not typically replicate in the host.
In one embodiment, a nucleic acid expression vector of this invention is administered in combination with one or more other nucleic acid vectors, as a xe2x80x9cnaked nucleic acid immunizationxe2x80x9d to protect against multiple viral diseases. In a specific embodiment for vaccinating birds, the other viral diseases can be avian polyomavirus, Pacheco""s disease virus, or psittacine beak and feather disease virus.
In another specific embodiment, the vaccine composition comprising an immunogenic amount of a C. psittaci major outer membrane protein (MOMP) polypeptide lacking variable regions VD1 and VD2 is administered in combination with one or more recombinant viral proteins from viruses that infect and cause disease in psittacine birds.
Any vaccine composition of this invention can further comprise an adjuvant suitable for use in the species to which the vaccine is to be administered, such as avian or mammalian species. Examples of such adjuvants include but are not limited to a cytokine, such as a lymphokine, a monokine or a chemokine, or a cytokine inducer or an agent that facilitates the entry of the antigen into the cytoplasm of the cell. Other examples of adjuvants that can useful in the present invention include but are not limited to plasmid DNA or bacterial agents. An adjuvant can also include, for example, immunomodulators and co-stimulatory molecules. Additional adjuvants include any compound described in Chapter 7 (pp 141-227) of xe2x80x98Vaccine Design, The Subunit and Adjuvant Approachxe2x80x99 (eds. Powell, M. F. and Newman, M. J.) Pharmaceutical Biotechnology, Volume 6, Plenum Press (New York).
Any vaccine composition of this invention can further comprise a pharmaceutically acceptable carrier. By xe2x80x9cpharmaceutically acceptablexe2x80x9d is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. A pharmaceutically acceptable carrier can comprise saline or other suitable carriers (Arnon, R., (Ed.) Synthetic Vaccines 1:83-92; CRC Press, Inc., Boca Raton, Fla. 1987).
Methods of Vaccination
The present invention further provides methods of vaccinating a subject to induce an immunological response capable of preventing a subsequent C. psittaci infection. The vaccine can be administered to an animal of the avian species, such as poultry, turkeys and companion birds; additionally, particularly for handlers of such avian species, the vaccines can be administered to mammals such as humans. In particular, birds which can be treated by the invention can be any of the various species of birds which are classified as being members of the Psittaciformes order. Examples of such birds include, but are not limited to, Budgerigars (Melopsittacus undulatus), caiques (e.g., Pionites leucogaster leucogaster), macaws (e.g., Ara ararauna), Amazon parrots (e.g., Amazona ochrocephala auropalliata, conures (e.g., Pyrrhara picta, Aratinga wagleri wagleri, Aratinga solstitialis, Aratinga guarouba, Aratinga holochlora rubritorquis or Aratinga acuticaudata acuticaudata), cockatoos (e.g., Cacatua moluccensis, Cacatua ducorps, Cacatua sulphura, Cacatua goffini or Cacatua alba), Splendid Parakeets (Neophema splendida), Pionus Parrots (Pionus maximillani), African Grey Parrots (Psittacus erithacus erithacus, Eclectus Parrots (Electus roratus), Cockatiels (Nymphicus hollandicus) and parakeets (e.g. Psittacula krameri krameri).
Thus, the present invention provides a method of preventing a Chlamydia psittaci infection in a subject comprising administering to the subject a vaccine comprising an immunogenic amount of a C. psittaci major outer membrane protein (MOMP) polypeptide lacking variable regions VD1 and VD2. In a specific embodiment, the method comprises an immunogenic amount of a MOMP polypeptide having an amino acid sequence as set forth in either SEQ ID NO:1 or SEQ ID NO:2. In another embodiment, the method comprises an immunogenic amount of an MBP-MOMP polypeptide fusion protein.
The present invention further provides a method of preventing a Chlamydia psittaci infection in a subject comprising administering to the subject a vaccine comprising an immunizing amount of a nucleic acid vector for the transient expression in a eukaryotic cell comprising a eukaryotic promoter functionally linked to a nucleic acid encoding a C. psittaci MOMP polypeptide lacking regions VD1 and VD2 of MOMP. In a specific embodiment, the method comprises a nucleic acid encoding an amino acid sequence as set forth in SEQ ID NO:7 or SEQ ID NO:8.
In a specific embodiment, the present invention provides a method of preventing a Chlamydia psittaci infection in a subject comprising administering to the subject a vaccine comprising an immunizing amount of a nucleic acid vector for transient expression in a eukaryotic cell comprising a cytomegalovirus promoter functionally linked to a nucleic acid having the nucleotide sequence set forth in either SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.
Vaccine compositions can be administered to a subject or an animal model by any of many standard means for administering the particular composition. For example, compositions can be administered orally, sublingually, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, topically, transdermally, or the like. Compositions can be administered, for example as a complex with cationic liposomes, encapsulated in anionic liposomes, or encapsulated in microcapsules. Compositions can include various amounts of the selected composition in combination with a pharmaceutically acceptable carrier and, in addition, if desired, may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, etc. Parental administration, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Actual methods of preparing dosage forms are known, or will be apparent, to those skilled in this art; for example, see Martin, E. W., Ed., Remington""s Pharmaceutical Sciences, latest edition, Mack Publishing Co., Easton, Pa.
In one embodiment, a vaccine of this invention, whether a protein or a nucleic acid vaccine, is administered on a regular booster schedule, for example, every six months, to companion birds of the order Psittaciformes. The vaccine may be advantageously administered to such birds orally, such as in pill form, or intranasally in a spray, or intraocularly in a drop. Alternatively, the vaccine may be administered intramuscularly or subcutaneously.
The following examples are intended to illustrate, but not limit, the invention. While they are typical of those that might be used, other procedures known to those skilled in the art may be alternatively employed.